Myelin is a multi-lamellar structure that surrounds axons in both the CNS and PNS facilitating nerve conduction. P0, a transmembrane glycoprotein of the immunoglobulin super family, is the major structural protein in PNS myelin, and is expressed exclusively in Sehwann cells, the myelinating glia. Human mutations in P0 give rise to peripheral demyelinating neuropathies. Our long range goal is to understand the role of P0 in myelination and disease. Several lines of evidence demonstrate that P0 acts as a homophilic adhesion molecule, suggesting that P0 mediates myelin compaction through homophilic interactions between adjacent membrane leaflets. Analysis of the crystal structure of the P0-extracelfular domain indicates that adjacent P0 molecules have the potential to interact in cis to form tetramers, and that these tetramers may further interact in trans to mediate homophilic adhesion. Furthermore, mutations of amino acid residues that the crystal structure identifies as critical to both cis and trans interactions cause a demyelinating peripheral neuropathy in patients. Mutations within the cytoplasmic domain of P0 are also found in patients with inherited demyelinating neuropathy and deletions within the cytoplasmic domain have been experimentally shown to result in loss of adhesive function. We have shown that point mutations of serine and threonine in a consensus Protein Kinase C binding site, as well as a second nearby serine, which corresponds to a human nonsense mutation giving rise to demyelinating neuropathy, are critical to adhesive function. Furthermore, our deletion analysis suggests that the juxtamembrane cytoplasmic region is also important for adhesive function. Thus both the extracellular and intracellular domains of P0 are necessary for homophilic adhesion in vitro and for myelination in vivoadhesion. P0 may also play a regulatory role during myelination. Complete loss of P0 in mice through homologous recombination not only produces a demyelinating peripheral neuropathy, but also markedly alters the pattern of myelin-specific gene expression in peripheral nerve. However, the relationship between adhesive function and this regulatory function is not known. In this proposal we will further define the regions and specific residues in the extracellular and intracellular domains that are essential for adhesive function through expression of mutated P0 in established cell lines and analysis of P0-mediated, as well as attempt to resolve the relationship between the seemingly separate P0 functions, adhesive and myelination.